CN114751971A - Paralichthys olivaceus male-related Dmrt1 recombinant protein and application thereof - Google Patents
Paralichthys olivaceus male-related Dmrt1 recombinant protein and application thereof Download PDFInfo
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- CN114751971A CN114751971A CN202210356947.0A CN202210356947A CN114751971A CN 114751971 A CN114751971 A CN 114751971A CN 202210356947 A CN202210356947 A CN 202210356947A CN 114751971 A CN114751971 A CN 114751971A
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- dmrt1
- recombinant protein
- recombinant
- paralichthys olivaceus
- protein
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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- C12N15/09—Recombinant DNA-technology
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Abstract
The invention belongs to the technical field of molecular biology, and particularly relates to a paralichthys olivaceus Dmrt1 recombinant protein and application thereof. The invention obtains the recombinant protein of the Paralichthys olivaceus Dmrt1 for the first time by using an in-vitro recombinant expression technology, provides a preparation method of the recombinant protein, and incubates the gonadal tissue of the Paralichthys olivaceus by using the recombinant protein, thereby showing that the recombinant protein has bioactivity. The invention injects the recombinant Dmrt1 protein into the abdominal cavity of the adult female paralichthys olivaceus to cause the condensation of the nuclear chromatin of the oocyte in the ovarian tissue. Further injection into the abdominal cavity of gonadal undifferentiated fry resulted in significant drop and rise in estrogen and androgen levels in the juvenile fish (p <0.05), respectively, and the male rate increased from 0% to 82%. The recombinant protein is shown to be used for sex control of fishes such as paralichthys olivaceus and the like, and a new method and a new way can be provided for sex control and parthenocarpic production in fish culture.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a paralichthys olivaceus Dmrt1 recombinant protein and application thereof.
Technical Field
Dmrt1(double and mab-3-related transcription factor 1) as a functional conserved factor was shown to activate the testis-specific gene and reduce the estrogen level in vivo, suggesting that it plays a key role in sex determination and testis differentiation in fish. The sequence of the dmrt1 gene has been obtained from various fishes, and mRNA transcription and expression during gonad differentiation and development has been studied, and as a result, it was suggested that the gene began to be expressed at an early stage of the testis differentiation and continued to be highly expressed during the differentiation, and researchers speculated that dmrt1 might be involved in the formation of male sex phenotype. Further gene knockout and overexpression studies have shown that the knockout of dmrt1 in model fish zebrafish affects the mitosis of the spermary spermatogonium, indicating that it is involved in the maintenance of germ cells, but does not achieve a complete conversion of males to females; overexpression of dmrt1 in another model medaka also did not result in female to male conversion; however, overexpression of dmrt1 in the freshwater economic fish nile tilapia leads to development of a spermary-like structure of the ovary of a genetic female individual, so that the gene is considered to be involved in the process of spermary differentiation, but only less than 5 percent of individuals are completely reversed. Therefore, the precise role of dmrt1 in fish male differentiation remains to be determined. Compared with mRNA, the protein is a direct performer of gene function and has higher correlation with biological phenotype in function, but the research on the protein level of Dmrt1 in fish gonad differentiation is mainly limited to zebrafish, medaka, Nile tilapia and the like, and the expression diagram of Dmrt1 protein is analyzed only by Western blot and immunohistochemistry, and the result indicates that the protein plays an important role in male differentiation. Transfection experiments with eukaryotic cells have shown that the Dmrt1 protein can directly or indirectly inhibit transcription of the estradiol synthesis key enzyme gene, cyp19a, but this study cannot clarify its function by observing whether Dmrt1 acts directly on male phenotype formation. The Dmrt1 recombinant protein can be obtained, so that the gene function can be directly researched, and the Dmrt1 recombinant protein can also be used as an inducer for controlling sex phenotype formation. At present, the in vitro protein expression vectors commonly used mainly comprise Blunt E2, pET and the like, and the recombination effect is different for different proteins.
At present, methods for inducing masculinization in fish mainly comprise exogenous androgen, estrogen receptor inhibitor and aromatase inhibitor, and although the methods can artificially regulate and control the sex transformation of the fish to obtain the functional male fish, the application of the methods has certain limitations. Sex hormone and receptor inhibitor thereof are not allowed to be used in cultivation because of polluting water environment; the aromatase inhibitor has potential biological influence due to relatively high use cost or accumulation of exogenous inducers in bodies, and is not suitable for application in culture production. In contrast, the recombinant protein of fish, as a novel inducer, can not only effectively control sex, but also avoid exogenous pollution, thus having potential application prospect.
Paralichthys olivaceus belongs to Pleuroectales, Paralichthyidae and Paralichthys, is a cold-warm benthic fish, has high economic value and is an important marine culture fish in China. The growth of the paralichthys olivaceus has obvious sex diphasic, the female growth is obviously faster than that of the male, and in order to realize full-female production and obtain higher economic benefit, the research on the sex formation mechanism of the female and the male is necessary. Therefore, the method for controlling sex of paralichthys olivaceus by utilizing sex-related gene function research has important application value in researching sex-related gene function of paralichthys olivaceus, and determining the function of paralichthys olivaceus in sex control, and further applying the method to artificial intervention of sex conversion for unisexual culture.
Disclosure of Invention
The invention aims to provide a paralichthys olivaceus male-related Dmrt1 recombinant protein and application thereof.
In order to realize the purpose, the invention adopts the technical scheme that:
a recombinant protein of male-related Dmrt1 of paralichthys olivaceus is obtained by inserting a Dmrt1 coding region into a pET-30a-EGFP vector, so that the recombinant protein of Dmrt1 of the paralichthys olivaceus is obtained.
The coding region of the nucleotide sequence XM _020107068.1 of the dmrt1 gene is shown in SEQ ID NO. 1.
Obtaining the Dmrt1 recombinant protein:
(1) performing PCR amplification by using the Paralichthys olivaceus testis cDNA as a template;
(2) purifying the PCR product, connecting the purified PCR product with an Escherichia coli expression vector pET-30a-EGFP, converting the connection product into Escherichia coli E.coli, sequencing and identifying a recombinant to obtain a recombinant plasmid pET-30a-EGFP-Dmrt 1;
(3) transferring the recombinant plasmid into E.coli Transetta (DE3) chemically competent cells, inoculating the cells into an auto-induction complex culture medium containing 0.1% kanamycin, and then carrying out induction culture, purification and renaturation to obtain the Dmrt1 recombinant protein.
The primer adopted by the PCR amplification in the step (1) is
Dmrt1-F：5’-AAGGCCATGGCTGATATGAACAAGGACAAGCAGAG-3’；
Dmrt1-R：5’-TGAATCGATACGCATATTGATGCCATCCACGTCGA-3’。
The activity of the recombinant protein is verified by performing flounder gonad pre-incubation by using an L15 culture medium containing 1% antibiotic, and then performing activity verification by using a recombinant Dmrt1 protein and Lipofectamine TM3000 fresh culture medium of transfection reagent was subjected to gonad incubation, and the expression change of downstream gene cyp19a was analyzed.
An application of a Dmrt1 recombinant protein related to male flounder, and an application of the Dmrt1 recombinant protein in sex control or parthenocarpy culture of the flounder.
The Dmrt1 recombinant protein caused nuclear chromatin condensation of oocytes in adult fish ovarian tissue, as well as changes in juvenile sex hormone levels and androgenic ratios.
The invention has the advantages that:
the invention obtains the Paralichthys olivaceus Dmrt1 recombinant protein by using an in vitro recombinant expression technology for the first time, and verifies the biological activity of the recombinant protein by using in vitro gonad tissue incubation. The flounder Dmrt1 recombinant protein can cause the condensation of nuclear chromatin of oocytes in ovarian tissues of the flounder through intraperitoneal injection of female adults; and the intraperitoneal injection of the juvenile fish in the differentiation period of the genetic female can respectively and remarkably reduce and increase the estrogen and androgen levels in vivo (p <0.05), and the male rate is increased from 0% to 82%, which indicates that the recombinant protein can be applied to sex control and parthenocarpy culture of paralichthys olivaceus.
The technical method can be used for the induction research of the gene on the male sex of the paralichthys olivaceus and can also be popularized and applied to other fishes.
Drawings
FIG. 1 shows SDS-PAGE detection of prokaryotic expression and purification of Dmrt1 protein obtained according to the present invention; wherein, A, protein expression (vector Blunt E2); b, protein expression (vector pET-30 a-EGFP); and C, protein purification (a vector pET-30 a-EGFP). Arrows indicate the protein of interest. M, marker; sNIUninduced supernatant; pNI(iv), uninduced precipitation; s, supernatant after induction; p, precipitation after induction; f, cleaning the mixed protein filtrate; 1-7, eluting the target protein.
FIG. 2 shows WB identification and cyp19a gene expression changes of obtained Dmrt1 recombinant protein incubated ovary and testis tissues; wherein, A and WB are identified; b, expression of cyp19a gene was varied. Blue box, result of Dmrt1 recombinant protein incubation; green box, result of EGFP protein incubation.
FIG. 3 shows the change of gonad histology of adult female flounder after intraperitoneal injection of the obtained Dmrt1 recombinant protein; among them, black circles, dysplastic cell morphology. Og, oogonia; oo, oocyte; bar, 5 μm.
FIG. 4 shows the sex hormone level change of the obtained Dmrt1 recombinant protein after intraperitoneal injection of the juvenile gynogenesis of paralichthys olivaceus;
FIG. 5 shows sex ratio and gonad histological changes of the obtained Dmrt1 recombinant protein after intraperitoneal injection of the flounder gynogenesis juvenile fish; wherein, A, the change of sex ratio; b (1), non-injected ovaries; (2) ovaries after EGFP injection; (3) ovaries injected with Dmrt1 recombinant protein; (4) and, the spermary of the recombinant protein was injected with Dmrt 1. Red circles, abnormal cell morphology. Og, oogonia; oo, oocytes; oc, ovarian cavity; sg, spermatogonial cells; so, spermatocytes; bar, 5 μm.
Detailed Description
The following further description of the embodiments of the present invention is provided in conjunction with the examples and the accompanying drawings, and it is to be understood that the embodiments described herein are merely for purposes of illustration and explanation and are not intended to limit the invention.
The recombinant expression plasmid of the turbot Dmrt1 constructed by the invention is induced and purified to obtain recombinant protein, the obtained protein is specifically a green fluorescent fusion protein obtained by inserting a Dmrt1 coding region into a specific pET-30a-EGFP carrier and connecting the two, the obtained protein can obviously improve the yield and the expression stability of the recombinant protein, and the tissue incubation result shows that the recombinant protein has biological activity, can be used for intraperitoneal injection of the turbot, can cause the high condensation of nucleus chromatin of oocytes in an ovary tissue of adult fish, and successfully changes the sex phenotype of juvenile fish.
Example 1 prokaryotic recombinant expression of Paralichthys olivaceus Dmrt1 protein
1. Construction of recombinant plasmid Blunt E2-Dmrt1 and recombinant strain
Extracting RNA of a paralichthys olivaceus testis tissue, carrying out reverse transcription to obtain cDNA, amplifying all coding regions of the paralichthys olivaceus Dmrt1 by using PCR (polymerase chain reaction), wherein the coded amino acid sequence is shown as SEQ ID NO. 1, and then using a designed specific primer, the sequence of which is as follows:
Dmrt1-F：5’-ATGAACAAGGACAAGCAGAG-3’；
Dmrt1-R：5’-ATTGATGCCATCCACGTCGA-3’
performing PCR amplification, performing gel recovery, purification, sequencing and verification on the amplified fragment, connecting the fragment with a linearized Blunt E2 vector, transforming a recombinant plasmid into escherichia coli DH5 alpha competence, performing overnight culture, selecting a monoclonal colony to perform bacterial liquid PCR amplification detection, and handing the colony to a biological technology (Beijing) of Borkhovy Rui for sequencing and identification.
The amplification conditions were:
pre-denaturation: 94 ℃ for 2 min; denaturation, annealing and extension: 30s at 94 ℃, 30s at 60 ℃ and 1min at 72 ℃ for 30s, for 35 cycles; final extension: 5min at 72 ℃.
The amino acid sequence of the SEQ ID NO. 1 is Dmrt 1:
the recombinant plasmid Blunt E2-Dmrt1 was transformed into E.coli Transetta (DE3), spread on LB solid medium containing 0.1% kanamycin, and cultured overnight at 37 ℃ in an inverted state. Then selecting a single colony to carry out PCR detection and identification on the bacteria liquid. And (3) carrying out electrophoresis on the PCR product through 1% agarose gel, observing the size of a target band of the amplified product under a gel imager, and taking a bacterial colony which is consistent with the size of the expected fragment as a recombinant strain.
2. Inducible expression of recombinant strains
2.1 inducible expression of recombinant strains: inoculating the recombinant strain on an LB solid culture medium plate containing 0.1% kanamycin in a streaking mode, carrying out inverted culture at 37 ℃ for 14-16h, selecting a single colony, inoculating the single colony on an LB liquid culture medium containing 0.1% kanamycin, and placing the single colony at 37 ℃ for culture at 200 r/min for 12-16h to serve as a primary seed; inoculating the first seed with 1% inoculum size in a culture medium containing 0.1% kanamycin, adding IPTG (0.5mM), inducing and culturing at 37 deg.C under 200 rpm for 16h, and collecting bacterial liquid.
2.2 collection and detection of Dmrt1 recombinant protein: the bacterial solution was centrifuged at 4,000g for 10min at 4 ℃ to collect the cells in PBS (137mM NaCl, 2.7mM KCl, 10mM Na)2HPO4，1.8mMKH2PO4pH 7.4), resuspension with bacterial lysate containing protease inhibitors, and 250W ultrasonication on ice for 25 min. Centrifuge at 10,000g for 15min at 4 ℃ and collect the precipitate. The Dmrt1 recombinant protein was analyzed by polyacrylamide gel electrophoresis (SDS-PAGE) (see FIG. 1A).
The expression yield of the recombinant protein obtained from the Blunt E2 vector is low, so that the recombinant protein cannot be further analyzed and applied, and the vector may not be suitable for use.
Example 2
1. Construction of recombinant plasmid pET-30a-EGFP-Dmrt1 and recombinant strain
Extracting RNA of a paralichthys olivaceus testis tissue, carrying out reverse transcription to obtain cDNA, amplifying all coding regions of the paralichthys olivaceus Dmrt1 by using PCR, wherein the coded amino acid sequence is shown as SEQ ID NO. 1, and then using a designed specific primer, the sequence of which is:
Dmrt1-F：5’-AAGGCCATGGCTGATATGAACAAGGACAAGCAGAG-3’；
Dmrt1-R：5’-TGAATCGATACGCATATTGATGCCATCCACGTCGA-3’
performing PCR amplification, performing gel recovery, purification, sequencing and verification on the amplified fragment, performing homologous recombination with a linearized pET-30a-EGFP vector, transforming a recombinant plasmid into escherichia coli DH5 alpha competence, performing overnight culture, selecting a monoclonal colony, performing bacterial liquid PCR amplification detection, and performing sequencing and identification by Miao Boxing Ke biotechnology (Beijing) Co.
The amplification conditions were:
pre-denaturation: 2min at 94 ℃; denaturation, annealing and extension: 30s at 94 ℃, 30s at 60 ℃ and 1min at 72 ℃ for 30s for 35 cycles; final extension: 5min at 72 ℃.
The amino acid sequence of the SEQ ID NO. 1 is Dmrt 1:
the recombinant plasmid pET-30a-EGFP-Dmrt1 was transformed into E.coli Transetta (DE3), spread on LB solid medium containing 0.1% kanamycin, and cultured overnight at 37 ℃ in an inverted state. Then selecting a single colony to carry out PCR detection and identification on the bacteria liquid. And (3) carrying out electrophoresis on the PCR product through 1% agarose gel, observing the size of a target band of the amplified product under a gel imager, and taking a bacterial colony which is consistent with the size of the expected fragment as a recombinant strain.
2. Inducible expression of recombinant strain and purification of Dmrt1 recombinant protein
2.1 inducible expression of recombinant strains: inoculating the recombinant strain on an LB solid culture medium plate containing 0.1% kanamycin in a streaking mode, carrying out inverted culture at 37 ℃ for 14-16h, selecting a single colony, inoculating the single colony on an LB liquid culture medium containing 0.1% kanamycin, and placing the single colony at 37 ℃ for culture at 200 r/min for 12-16h to serve as a primary seed; inoculating the first-stage seeds in an auto-induction compound culture medium containing 0.1% kanamycin in an inoculation amount of 1%, culturing at 37 ℃ at 200 rpm for 16h, and collecting bacterial liquid.
2.2 collection and purification of Dmrt1 recombinant protein: centrifuging the bacterial liquid at 4,000g for 10min at 4 ℃, collecting thalli, washing for 3 times by PBS, carrying out heavy suspension by using bacterial lysate containing protease inhibitor, and carrying out ultrasonic lysis for 25min at 250W on ice. Centrifuge at 10,000g for 15min at 4 ℃ and collect the precipitate.
The protein pellet was resuspended in 10mL of equilibration buffer (5mM imidazole, 0.5mM NaCl, 8M urea, 20mM Tris-HCl, pH 7.9) per g of protein pellet, solubilized by vortexing at room temperature for 10min, and centrifuged at 10,000g at 4 ℃ for 20min to collect the supernatant. The supernatant was loaded on a column (Ni)2+Column), washed with 15 column volumes of equilibration buffer, and washed free of contaminating proteins. The elution was carried out using an appropriate amount of elution buffer (500mM imidazole, 0.5mM NaCl, 8M urea, 20mM Tris-HCl, pH 7.9), and the elution peak was collected.
Dialytic renaturation of Dmrt1 recombinant protein
The purified protein was placed in an MD34(Mw50,000) dialysis bag and soaked at a volume ratio of 1:100 between the protein sample and the dialysis buffer, the main components of the dialysis buffer were 50mM Tris, 24mM NaCl, 1mM KCl, 0.9mM L-glutathione reduction (GSH), 0.1mM L-glutathione oxidation (GSSG) and imidazole at different concentrations (250mM, 100mM, 0 mM). The dialysis time for each gradient was 6h, the last gradient was repeated 2 times, and the dialyzed protein was stored at-80 ℃. The Dmrt1 recombinant protein and the non-induced recombinant bacterial protein after dialysis were analyzed by SDS-PAGE, and the molecular weights of the proteins were determined (FIG. 1B, C), which were consistent with the expected protein sizes. This indicates that the Dmrt1 recombinant protein exists in the form of insoluble inclusion bodies and that the recombinant protein is obtained in higher purity after purification. Thus obtaining the flounder Dmrt1 recombinant protein, wherein the protein length is as follows: 302 amino acids, type: amino acids, chain type: single strand, properties: the predicted molecular weight is 33.0kDa, isoelectric point is 6.30, with conserved DM and Pfam domains.
Example 3Dmrt1 in vitro Activity verification of recombinant proteins
Tissue incubation of Dmrt1 recombinant proteins
The recombinant purified Paralichthys olivaceus Dmrt1 protein of example 2 was diluted to 1000. mu.g/mL in PBS as described above.
Collecting gonadal tissue of 4 tails of each male and female adult wild-type Paralichthys olivaceus, repeatedly washing with PBS containing 1% penicillin (10kU/mL) -streptomycin (10mg/mL) mixed antibiotic, and cutting into about 1.0mm3Is placed in a 24-well plate and preincubated with L15 medium containing 1% antibiotic at 25 ℃ for 6h, followed by recombinant protein Dmrt1 (100. mu.g) and Lipofectamine containing recombinant purified Dmrt1 of example 2TM3000 fresh medium of transfection reagent was incubated at 25 ℃ for 24h and the incubated samples were stored at-80 ℃ until use.
WB and qPCR analysis
A part of the samples were extracted for total protein by Radioimmunoprecipitation (RIPA), added with 5 Xloading buffer, and subjected to boiling water bath for 8-10min for WB detection. The remaining sample was subjected to Trizol to extract total RNA, and the concentration and purity of RNA were checked using a NanoDrop 2000 spectrophotometer and 1.5% agarose gel electrophoresis. The kit is used for synthesizing first strand cDNA by reverse transcription, and the first strand cDNA is used for qPCR analysis of gene expression quantity.
The results show that recombinant proteins can enter gonadal tissues by means of in vitro transfection reagents, see fig. 2A. After the incubation of the Dmrt1 recombinant protein, the expression level of the cyp19a gene in both ovary and testis was significantly reduced (p <0.05), as shown in FIG. 2B. The result shows that the purified Dmrt1 recombinant protein can remarkably inhibit the expression of cyp19a in gonads (p <0.05) and has biological activity.
Example 4 Effect of Dmrt1 recombinant protein on adult ovarian development of Paralichthys olivaceus
Intraperitoneal injection of Dmrt1 recombinant protein into female adult
Selecting 20 healthy wild adult flounder female fish, injecting 100 mu g of the recombinant purified protein in the embodiment 2 per g of body weight, simultaneously adding in vivo-jetPEI transfection reagent, injecting once every 3d, continuously injecting for 3 times, and culturing for 21 d.
2. Histological observation
Ovaries were removed, placed in davidsos fixative and histologic changes were observed using tissue sections. The experimental group was injected with Dmrt1 recombinant protein and the control group was injected with EGFP protein.
The results show that after the injection of the Dmrt1 recombinant protein, the ovary of 50% individuals is abnormal compared with the control group, as shown in figure 3, the oocyte nuclear chromatin of the ovary is condensed, which indicates that the Dmrt1 has a certain inhibiting effect on the development of the adult ovary.
Example 5 Effect of Dmrt1 recombinant proteins on the formation of sex phenotype of Paralichthys olivaceus
Intraperitoneal injection of genetic female juvenile fish of Dmrt1 recombinant protein
Healthy hereditary female (gynogenesis) flounder juvenile fish with a total length of 1.2-1.5cm are randomly divided into 3 groups: a Dmrt1 recombinant protein injection group, an EGFP protein injection group and an uninjected group. Dmrt1 recombinant protein and EGFP protein were transfected every 7d with in vivo-jetPEI transfection reagent. The injection concentration is 10 mug/tail when the full length is 1.2-1.5cm, then the concentration is increased proportionally with the growth of the juvenile fish, when the full length is 7.0cm, the concentration is increased to 100 mug/tail, the injection is stopped, and the cultivation is continued.
2. Sex hormone determination and histological observation
A sample of 10.0cm in length was collected, the head, trunk and visceral mass were removed, and the remaining gonadal sites were used for sex hormone determination, and levels of 17 β -estradiol (17 β -E2), testosterone (T) and 11-ketotestosterone (11-KT) were determined using an enzyme-linked immunosorbent assay (ELISA) kit. Randomly selecting 20 pieces, and observing the sex ratio and the histological characteristics by using the tissue slices.
The results showed that 17 β -E2 was at a lower level (p <0.05) in gonads, while T and 11-KT were at higher levels (p <0.05) after injection of Dmrt1 recombinant protein compared to EGFP and non-injected groups, as shown in FIG. 4. The female rate was 100% for both EGFP protein treated and non-injected groups, while the female rate was 18% (male rate was 82%) for Dmrt1 recombinant protein treated groups, as shown in fig. 5A. The EGFP protein-treated group and the non-injected group had normal development of ovaries, the Dmrt1 recombinant protein-treated group had normal development of spermary, the ovaries were abnormal, and the nuclear chromatin of oocytes in the ovaries was highly condensed, as shown in fig. 5B.
The incubation of the paralichthys olivaceus gonad tissue shows that the Dmrt1 recombinant protein can inhibit the expression of cyp19a and has biological activity. The result of an intraperitoneal injection experiment of female adult fish shows that the Dmrt1 recombinant protein can cause the condensation of nuclear chromatin of oocytes in ovaries and has a certain inhibiting effect on the development of adult ovaries. Further genetic female paralichthys olivaceus juvenile intraperitoneal injection experiments show that the change of female and androgen levels in juvenile fish can be influenced after the Dmrt1 recombinant protein is injected, so that the sex phenotype of the juvenile fish is changed, and a new biological insight and a new method are provided for parthenogenesis in aquaculture.
The above description is only a preferred embodiment of the present invention, and should not be taken as limiting the scope of the present invention, therefore, the appended claims should be construed as covering all equivalent variations of the present invention.
Sequence listing
<110> oceanic research institute of Chinese academy of sciences
<120> Paralichthys olivaceus male-related Dmrt1 recombinant protein and application thereof
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Lys Arg Phe Cys Asn Trp Arg Asp Cys Glu Cys Val Lys Cys Lys Leu
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Phe Ser Val Asp Gly Arg Ser Pro Thr Pro Thr Gly Ser Ala Ser Ala
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Ser Ser Leu Ala Phe Thr Gly Ser Arg Ser Ala Ser Ser Ser Ser Pro
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Ser Ala Gly Val Arg Ala His Ala Glu Gly Ala Ser Asp Leu Leu Met
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Glu Thr Ser Tyr Tyr Asn Phe Tyr Gln Pro Ser Cys Tyr Pro Thr Tyr
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Tyr Ser Asn Leu Tyr Asn Tyr Gln Gln Tyr Gln Gln Met Ser His Ser
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Asp Ser Arg Leu Ser Ser His Asn Met Ser Ser Pro Tyr Cys Met His
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Ser Tyr Tyr Pro Ala Ala Thr Tyr Leu Thr Gln Gly Leu Gly Ser Thr
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Asn Asn Tyr Ser Asp Thr Met Ala Ala Ser Phe Ser Pro Ser Asn Val
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Thr Ala Ala His Asp Pro Thr Met Thr Cys Arg Ser Ile Ser Ser Leu
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Claims (5)
1. A flounder male-related Dmrt1 recombinant protein, comprising: the recombinant protein is obtained by inserting a Dmrt1 coding region into a pET-30a-EGFP vector, thereby obtaining a paralichthys olivaceus Dmrt1 recombinant protein.
2. The paralichthys olivaceus male-related Dmrt1 recombinant protein of claim 1, wherein said recombinant protein comprises: the coding region of the nucleotide sequence XM _020107068.1 of the dmrt1 gene.
3. The flounder male-related Dmrt1 recombinant protein of claim 1 or 2, wherein said recombinant protein is selected from the group consisting of:
(1) performing PCR amplification by using the Paralichthys olivaceus testis cDNA as a template;
(2) the PCR product is purified and then connected with an escherichia coli expression vector pET-30a-EGFP, the connection product is converted into escherichia coli, and a recombinant is sequenced and identified to obtain a recombinant plasmid pET-30a-EGFP-Dmrt 1;
(3) transferring the recombinant plasmid into E.coli Transetta (DE3) chemically competent cells, inoculating the cells into an auto-induction complex culture medium containing 0.1% kanamycin, and then carrying out induction culture, purification and renaturation to obtain the Dmrt1 recombinant protein.
4. The paralichthys olivaceus male-related Dmrt1 recombinant protein of claim 3, wherein: the primer adopted by the PCR amplification in the step (1) is
Dmrt1-F：5’-AAGGCCATGGCTGATATGAACAAGGACAAGCAGAG-3’；
Dmrt1-R：5’-TGAATCGATACGCATATTGATGCCATCCACGTCGA-3’。
5. The use of the recombinant protein Dmrt1 related to male flounder of claim 1, wherein the recombinant protein comprises the following components: the Dmrt1 recombinant protein is applied to sex control or parthenocarpy culture of paralichthys olivaceus.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102355814A (en) * | 2009-02-08 | 2012-02-15 | 墨尔本大学 | Sex-determination and methods of specifying same |
| CN104630133A (en) * | 2015-02-05 | 2015-05-20 | 中国科学院海洋研究所 | Establishment method of Paralichthys olivaceus spermary cell line |
| JP2019154367A (en) * | 2018-03-15 | 2019-09-19 | 国立大学法人 鹿児島大学 | Transsexual induction method of fishes, transsexual induction device of fishes and sex control method of fishes |
| CN116602243A (en) * | 2023-05-12 | 2023-08-18 | 中国科学院海洋研究所 | Application of paralichthys olivaceus 17 beta-hydroxysteroid dehydrogenase 12b |
-
2022
- 2022-03-31 CN CN202210356947.0A patent/CN114751971B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102355814A (en) * | 2009-02-08 | 2012-02-15 | 墨尔本大学 | Sex-determination and methods of specifying same |
| US20120084873A1 (en) * | 2009-02-08 | 2012-04-05 | Andrew Sinclair | Sex-determination and methods of specifying same |
| CN104630133A (en) * | 2015-02-05 | 2015-05-20 | 中国科学院海洋研究所 | Establishment method of Paralichthys olivaceus spermary cell line |
| JP2019154367A (en) * | 2018-03-15 | 2019-09-19 | 国立大学法人 鹿児島大学 | Transsexual induction method of fishes, transsexual induction device of fishes and sex control method of fishes |
| CN116602243A (en) * | 2023-05-12 | 2023-08-18 | 中国科学院海洋研究所 | Application of paralichthys olivaceus 17 beta-hydroxysteroid dehydrogenase 12b |
Non-Patent Citations (5)
| Title |
|---|
| CHANG SHU等: "Function of Foxl2 and Dmrt1 proteins during gonadal differentiation in the olive flounder Paralichthys olivaceus", vol. 215 * |
| GENBANK: "PREDICTED: doublesex- and mab-3-related transcription factor 1 isoform X3 [Paralichthys olivaceus]", 《GENBANK》, pages 019962627 * |
| GENBANK: "PREDICTED: doublesex- and mab-3-related transcription factor 1 isoform X3 [Paralichthys olivaceus]:PREDICTED: doublesex- and mab-3-related transcription factor 1 isoform X3 [Paralichthys olivaceus]", 《GENBANK》, pages 0201 * |
| MING YING等: "Nuclear import of human sexual regulator DMRT1 is mediated by importin-β", 《BIOCHIMICA ET BIOPHYSICA ACTA (BBA) - MOLECULAR CELL RESEARCH》, vol. 1773, no. 6, pages 804 - 813 * |
| 文爱韵等: "牙鲆dmrt1基因的克隆及其与P450arom基因的组织表达分析", 《海洋科学》, vol. 34, no. 11 * |
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| CN114751971B (en) | 2023-10-20 |
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